We propose to express recombinant N- and C-terminal half-molecules of human serum transferrin and hen ovotransferrin in tissue cultures of baby hamster kidney cells in sufficient quantities for cellular function studies, nuclear magnetic resonance spectroscopy and X-ray crystallography. Functional studies will address a) the ability of the half-molecules to bind specifically metal ions and synergistic metal ions and b) the relative abilities of the human holo-transferrin, its half-molecules and a mixture of the half-molecules to bind and donate iron to HeLa cells in culture and to support cell division and growth. Site-directed mutagenesis of amino acids known and suspected to serve as ligands in transferrin to metal ions and anions will allow us to test the structural hypotheses regarding the function of these amino acid side chains and to corroborate the functional changes in naturally occurring mutants. Substitution of isotope-enriched amino acids, e.g., ring-deuterated HIs, Phe, Tyr, Trp, 15-N Arg, His, Lys, and 19-F substituted Phe, Tyr and Trp in the recombinant transferrin half-molecules will allow the partial resolution of the NMR spectra and "wild type" and mutated proteins. X-ray crystallographic studies of the recombinant transferrin half- molecules will be undertaken with the aim of delineating and refining the 3-dimensional structure of these proteins. Further studies of site-mutated proteins will be undertaken if the data warrant. Continuing studies of ovotransferrin from egg white and transferrin from human serum will be directed toward assigning NMR resonances associated with the binding sites to specific amino acid residues in the primary sequence. Primary methods will involve chemical modification of amino acid side chains in apo- and iron-proteins, proteolytic cleavage of the modified proteins and isolation of modified peptides by FPLC and HPLC. Alterations in the NMR spectra will be correlated with the structural information obtained.